Use of chemically inducible systems for transgene expression is a crucial requirement for modern plant biology research, as it allows (1) expression of transgenes that compromise plant viability or fertility when constitutively expressed and (2) spatiotemporal control of transgene expression levels. We describe the stringently regulated and highly responsive dexamethasone-inducible gene expression system pOp6/LhGR, which comprises the chimeric transcription activator LhGR and the corresponding pOp6 promoter. Upon induction, the LhGR activator binds to the pOp6 promoter and induces expression of the target gene of interest. We provide detailed protocols for inducing transgene expression at different developmental stages and in different plant species and discuss dexamethasone stability and use of its analogs. We also introduce new, versatile, GATEWAY-compatible binary vectors that are now available for the pOp6/LhGR system. © 2019 by John Wiley & Sons, Inc.

• Klíčová slova
• chemically inducible gene expression, dexamethasone, glucocorticoid,
• MeSH
• aktivace transkripce * účinky léků   MeSH
• Arabidopsis genetika   MeSH
• dexamethason farmakologie   MeSH
• DNA vazebné proteiny genetika   MeSH
• genetické techniky MeSH
• geneticky modifikované rostliny genetika   MeSH
• lac represory genetika   MeSH
• promotorové oblasti (genetika) MeSH
• proteiny z Escherichia coli genetika   MeSH
• receptory glukokortikoidů genetika   MeSH
• Saccharomyces cerevisiae - proteiny genetika   MeSH
• transgeny * MeSH
• transkripční faktory genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• dexamethason MeSH
• DNA vazebné proteiny MeSH
• GAL4 protein, S cerevisiae MeSH Prohlížeč
• lac represory MeSH
• LacI protein, E coli MeSH Prohlížeč
• proteiny z Escherichia coli MeSH
• receptory glukokortikoidů MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• transkripční faktory MeSH

BACKGROUND: Nitric oxide (NO) is an important mediator of physiologic processes in the airways; it plays a significant role in the regulation of the T helper type 1/type 2 balance and contributes to the development of atopic diseases. OBJECTIVE: We analysed several polymorphisms mainly in the promoter region of the inducible NO synthase (NOS2, iNOS) gene and investigated their associations with asthma and/or atopic phenotypes. METHODS: We performed a case-control study in 994 subjects (661 patients with atopic disorders, with subgroups of 304 patients with allergic asthma, and 333 healthy individuals), matched for sex, living in the same geographical area. Screening for polymorphisms was performed by combination of PCR and direct sequencing analysis. RESULTS: We analysed 14 nucleotide sequence variants, seven most common of which were typed in quite large groups of our asthmatic, atopic and control populations. None of these seven frequent polymorphisms was associated with the phenotype bronchial asthma or other atopic diseases. Nevertheless, three from six common promoter polymorphisms showed a significant relation to feather's positivity (P value from 0.01 to 0.03) and the NOS2 608Leu variant was significantly associated with asthma severity [p(corr) = 0.0005; odds ratio (OR) = 5.00, 95% confidence interval (CI): 1.88-13.33]. In haplotype analysis, the most common -2447C/-1659C/-1026G/-0.7del/-277A/Ser608 haplotype was associated with a lower risk of asthma when compared with the common haplotypes with frequency more than 5% (P = 0.01, p(corr) < 0.05; OR = 0.65, 95% CI: 0.56-0.77). CONCLUSION: Our findings suggest that inducible NOS can play a role in atopic disorders, and several polymorphisms in its gene may be important for asthma protection or susceptibility.

• MeSH
• bronchiální astma enzymologie   genetika   MeSH
• časná přecitlivělost enzymologie   genetika   MeSH
• dospělí MeSH
• genetická predispozice k nemoci MeSH
• genotyp MeSH
• haplotypy MeSH
• lidé středního věku MeSH
• lidé MeSH
• polymorfismus délky restrikčních fragmentů MeSH
• polymorfismus genetický * MeSH
• promotorové oblasti (genetika) * MeSH
• rozdělení chí kvadrát MeSH
• sekvenční analýza DNA MeSH
• studie případů a kontrol MeSH
• synthasa oxidu dusnatého, typ II genetika   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• synthasa oxidu dusnatého, typ II MeSH

An essential prerequisite for a controlled transgene expression is the choice of a suitable promoter. In the model diatom Phaeodactylum tricornutum, the most commonly used promoters for trans-gene expression are the light dependent lhcf1 promoters (derived from two endogenous genes encoding fucoxanthin chlorophyll a/c binding proteins) and the nitrate dependent nr promoter (derived from the endogenous nitrate reductase gene). In this study, we investigated the time dependent expression of the green fluorescent protein (GFP) reporter under control of the nitrate reductase promoter in independently genetically transformed P. tricornutum cell lines following induction of expression by change of the nitrogen source in the medium via flow cytometry, microscopy and western blotting. In all investigated cell lines, GFP fluorescence started to increase 1 h after change of the medium, the fastest increase rates were observed between 2 and 3 h. Fluorescence continued to increase slightly for up to 7 h even after transfer of the cells to ammonium medium. The subsequent decrease of GFP fluorescence was much slower than the increase, probably due to the stability of GFP. The investigation of several cell lines transformed with nr based constructs revealed that, also in the absence of nitrate, the promoter may show residual activity. Furthermore, we observed a strong variation of gene expression between independent cell lines, emphasising the importance of a thorough characterisation of genetically modified cell lines and their individual expression patterns.

• Klíčová slova
• Flow cytometry, Fluorescence intensity, Green fluorescent protein, Inducible promoter, Nitrate, Nitrogen source,
• Publikační typ
• časopisecké články MeSH

In order to construct plasmids bearing inducible high-copy-number phenotype, the cloning plasmid pBR322 was modified as follows: a DNA fragment containing a strong synthetic promoter (P1), synthetic lac operator (O1), DNA sequence corresponding to the RNAI/RNAII region of the Co1E1 replicon and the CAT gene transcription terminator was substituted for the 29 bp EcoRI/HindIII DNA fragment. Two types of plasmids were constructed in this way, differing in the orientation of the RNAI/RNAII fragment. Depending on the orientation these plasmids coded for RNA molecules representing either RNAI or RNAII domains. It was found that when RNAII molecules were overproduced the plasmid copy number was about 4 times higher than that of pBR322 and only negligible change in the plasmid copy-number value was observed upon overproduction of RNAI molecules.

• MeSH
• bakteriální RNA biosyntéza   MeSH
• bakteriální transformace MeSH
• bakteriocinové plazmidy * MeSH
• DNA bakterií MeSH
• fenotyp MeSH
• klonování DNA MeSH
• lac operon MeSH
• promotorové oblasti (genetika) MeSH
• regulace genové exprese u bakterií * MeSH
• replikon MeSH
• terminátorové oblasti (genetika) MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální RNA MeSH
• DNA bakterií MeSH

It has been well recognized that the promoter polymorphisms of interleukin-18 (IL-18) influence the level of cytokine expression. In our previously published data, we showed constitutive IL-18 expression in the epithelium of renal distal tubules in patients after kidney transplantation and significantly elevated IL-18 expression during acute rejection. In this study, we evaluated the clinical significance of two functional promoter polymorphisms of the IL-18 gene at positions -607 A/C (rs1946518) and -137 C/G (rs187238) in patients after kidney transplantation and looked for associations with the onset of graft function and the incidence of rejection episodes. Promoter polymorphisms in 124 patients and 103 unrelated controls were evaluated by sequence-specific primer polymerase chain reaction and the allele, genotype and haplotype frequencies were statistically correlated. We found a statistically different distribution of the allele frequency of -607 A/C polymorphism between patients with immediate or delayed onset of kidney graft function. Data showed that the C allele, which contributes to higher IL-18 expression, is more frequent in patients with delayed onset of function (P = 0.03, odds ratio = 1.93; 95% confidence interval = 1.15-3.25). A/C single nucleotide polymorphisms of the IL-18 promoter at position -607 may influence the onset of early kidney allograft function.

• MeSH
• časové faktory MeSH
• distální tubuly ledvin metabolismus   MeSH
• epitelové buňky metabolismus   MeSH
• homologní transplantace MeSH
• interleukin-18 biosyntéza   genetika   MeSH
• jednonukleotidový polymorfismus * MeSH
• lidé středního věku MeSH
• lidé MeSH
• obnova funkce genetika   MeSH
• přežívání štěpu genetika   MeSH
• promotorové oblasti (genetika) genetika   MeSH
• regulace genové exprese genetika   MeSH
• rejekce štěpu genetika   metabolismus   MeSH
• retrospektivní studie MeSH
• transplantace ledvin * MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• interleukin-18 MeSH

The 5'-end of the duck beta A-globin promoter contains a protein binding site BS-3/Sp1, the A + T-rich part of which could be involved in DNA bending. Plasmids were constructed using the pBend2 plasmid containing BS-3/Sp1. Circular permutation analysis of the fragments cut out from the plasmids using various restriction endonucleases, in the presence of a partially purified protein extract from embryonic duck erythrocytes, was performed. The results indicate that a rather complicated change in the fragment shape takes place upon protein binding, which is best explained as an induction of two points of bending or flexure within the fragment. Analogical points of flexure may exist at the protein-binding sites of the duck and chicken beta A-globin promoters in spite of differing DNA sequences.

• MeSH
• DNA vazebné proteiny metabolismus   MeSH
• globiny genetika   MeSH
• kachny genetika   MeSH
• konformace nukleové kyseliny * MeSH
• molekulární sekvence - údaje MeSH
• promotorové oblasti (genetika) genetika   MeSH
• sekvence nukleotidů MeSH
• transkripční faktor Sp1 MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• globiny MeSH
• transkripční faktor Sp1 MeSH

Francisella tularensis is a Gram-negative intracellular pathogen causing tularemia. A number of its potential virulence factors have been identified, but their biology and functions are not precisely known. Understanding the biological and immunological functions of these proteins requires adequate genetic tools for homologous and heterologous expression of cloned genes, maintaining both original structure and post-translational modifications. Here, we report the construction of a new multipurpose shuttle plasmid - pEVbr - which can be used for high-level expression in F. tularensis. The pEVbr plasmid has been constructed by modifying the TetR-regulated expression vector pEDL17 (LoVullo, 2012) that includes (i) a strong F. tularensis bfr promoter, and (ii) two tet operator sequences cloned into the promoter. The cloned green fluorescent protein (GFP), used as a reporter, demonstrated almost undetectable basal expression level under uninduced conditions and a highly dynamic dose-dependent response to the inducer. The utility of the system was further confirmed by cloning the gapA and FTT_1676 genes into the pEVbr vector and quantifying proteins expression in F. tularensis LVS, as well as by studying post-translational modification of the cloned genes. This study demonstrates that high levels of recombinant native-like Francisella proteins can be produced in Francisella cells. Hence, this system may be beneficial for the analysis of protein function and the development of new treatments and vaccines.

• Klíčová slova
• ATc inducible, Expression plasmid, Francisella tularensis, Regulated bfr promoter,
• MeSH
• Francisella tularensis * genetika   MeSH
• lidé MeSH
• plazmidy genetika   MeSH
• rekombinantní proteiny genetika   MeSH
• tetracyklin farmakologie   MeSH
• tularemie * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• rekombinantní proteiny MeSH
• tetracyklin MeSH

Non-canonical secondary structures in DNA are increasingly being revealed as critical players in DNA metabolism, including modulating the accessibility and activity of promoters. These structures comprise the so-called G-quadruplexes (G4s) that are formed from sequences rich in guanine bases. Using a well-defined transcriptional reporter system, we sought to systematically investigate the impact of the presence of G4 structures on transcription in yeast Saccharomyces cerevisiae. To this aim, different G4 prone sequences were modeled to vary the chance of intramolecular G4 formation, analyzed in vitro by Thioflavin T binding test and circular dichroism and then placed at the yeast ADE2 locus on chromosome XV, downstream and adjacent to a P53 response element (RE) and upstream from a minimal CYC1 promoter and Luciferase 1 (LUC1) reporter gene in isogenic strains. While the minimal CYC1 promoter provides basal reporter activity, the P53 RE enables LUC1 transactivation under the control of P53 family proteins expressed under the inducible GAL1 promoter. Thus, the impact of the different G4 prone sequences on both basal and P53 family protein-dependent expression was measured after shifting cells onto galactose containing medium. The results showed that the presence of G4 prone sequences upstream of a yeast minimal promoter increased its basal activity proportionally to their potential to form intramolecular G4 structures; consequently, this feature, when present near the target binding site of P53 family transcription factors, can be exploited to regulate the transcriptional activity of P53, P63 and P73 proteins.

• Klíčová slova
• G-quadruplex, p53, transcriptional activity, yeast,
• MeSH
• DNA metabolismus   MeSH
• G-kvadruplexy * MeSH
• nádorový supresorový protein p53 genetika   MeSH
• promotorové oblasti (genetika) MeSH
• Saccharomyces cerevisiae * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• nádorový supresorový protein p53 MeSH

Some metallo-supramolecular helical assemblies with size, shape, charge and amphipathic architectures similar to short cationic α-helical peptides have been shown to target and stabilise DNA G-quadruplexes (G4s) in vitro and downregulate the expression of G4-regulated genes in human cells. To expand the library of metallohelical structures that can act as efficient DNA G4 binders and downregulate genes containing G4-forming sequences in their promoter regions, we investigated the interaction of the two enantiomeric pairs of asymmetric Fe(II) triplex metallohelices with a series of five different DNA G4s formed by the human telomeric sequence (hTelo) and in the promoter regions of c-MYC, c-KIT, and k-RAS oncogenes. The metallohelices display preferential binding to G4s over duplex DNA in all investigated G4-forming sequences and induced arrest of DNA polymerase on template strands containing G4-forming sequences. Moreover, the investigated metallohelices suppressed the expression of c-MYC and k-RAS genes at mRNA and protein levels in HCT116 human cancer cells, as revealed by RT-qPCR analysis and western blotting.

• Klíčová slova
• DNA synthesis, G-quadruplexes, Metallohelices, expression of oncogenes, telomeres,
• MeSH
• DNA chemie   MeSH
• G-kvadruplexy * MeSH
• lidé MeSH
• nádory * MeSH
• onkogeny MeSH
• promotorové oblasti (genetika) MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA MeSH

In preeclampsia the cytotrophoblast invasion of the decidual vessels is reduced. The endothelia in the decidual vessels may influence cytotrophoblast invasion and remodeling of decidual spiral arteries. The decidual endothelial cells from preeclamptic placentas produce less matrix metalloproteinase-1 (MMP1) than those from normal placentas. MMPs form a group of enzymes that are capable of degrading components of extracellular matrix. The present study investigated the prevalence and possible association of an insertion of guanine in the promoter of the MMP1 gene in pregnancy-induced hypertension, preeclampsia and eclampsia in the Czech population. This was a case-control study. No differences were observed in genotype frequencies between cases and controls. The insertion of the guanine in the promoter of the MMP1 gene does not appear to increase the risk of development of pregnancy-induced hypertension, preeclampsia and eclampsia.

• MeSH
• dospělí MeSH
• frekvence genu genetika   MeSH
• genetická predispozice k nemoci genetika   MeSH
• genotyp MeSH
• hypertenze etiologie   genetika   MeSH
• jednonukleotidový polymorfismus genetika   MeSH
• kardiovaskulární komplikace v těhotenství * MeSH
• lidé MeSH
• matrixová metaloproteinasa 1 genetika   MeSH
• mladiství MeSH
• polymerázová řetězová reakce MeSH
• preeklampsie genetika   MeSH
• promotorové oblasti (genetika) genetika   MeSH
• rizikové faktory MeSH
• studie případů a kontrol MeSH
• těhotenství MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mladiství MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• matrixová metaloproteinasa 1 MeSH